The hypothalamic-pituitary-gonadal (HPG) axis regulates puberty initiation and reproductive function. This axis is active during the embryonic and neonatal stages of human life but then suppressed during childhood, then ultimately re-activated, first detected as an increase in amplitude and frequency of gonadotropin-releasing hormone (GnRH) pulses, to initiate puberty. The re-emergence of pulsatile GnRH release leads to increases in the secretion of the pituitary gonadotropins, luteinizing hormone (LH) and follicle stimulating hormone (FSH), and consequent activation of gonadal function. Pubertal timing is influenced by complex interactions of genetic, nutritional, environmental and socioeconomic factors. Early activation of the HPG axis results in gonadotropin- dependent or central precocious puberty (CPP), clinically defined by the development of secondary sexual characteristics before age 8 years in girls and 9 years in boys. The timing of puberty is associated with risks of subsequent disease; earlier age of menarche in girls is associated with increased risks of breast cancer, endometrial cancer, obesity, type 2 diabetes and cardiovascular disease. The genetic determinants of the timing of human pubertal development and in particular CPP are largely unknown. We recently identified loss- of-function mutations in the imprinted MKRN3 gene, encoding makorin ring finger protein 3, in patients with CPP, using whole exome sequencing. Mkrn3is highly expressed in the arcuate nucleus of juvenile mice, with a marked reduction prior to puberty onset. Based on these data, we hypothesize that MKRN3 acts as a brake for GnRH release during the dormant prepubertal phase, the first peptide identified to have such an inhibitory role. However, little is known about the function of this protein. The long-term goal of this project is to elucidate the molecular, cellular, and physiologic mechanisms by which MKRN3 controls the timing of puberty onset, and by which MKRN3 mutations result in early activation of the central reproductive axis. In the first aim, we will identify novel MKRN3 defects in patients with CPP and expand our understanding of the phenotype of patients with MKRN3 deficiency. In the second aim, we will determine the reproductive role of Mkrn3 in vivothrough a series of expression and functional studies in mice with and without functional Mkrn3. In the third aim, we will identify MKRN3 partners and/or targets in vitro by performing capture proteomics in a neuronal cell line as well as comparing gene expression profiles in neuronal progenitor cells derived from human induced pluripotent stem cells with and without functional MKRN3. The successful completion of these aims will help us to understand the actions of MKRN3, a novel regulator of GnRH re-activation, in the neuroendocrine control of pubertal timing and may also have a broader role in neuronal differentiation, development and maturation. A complete understanding of the role of MRKN3 in GnRH regulation may also identify novel factors involved in the neuroendocrine control of reproduction, and lead to the development of new tools for the management of reproductive disorders.